Abstract Significance: ImmuoPET is a powerful in vivo imaging technique with unique advantages over standard of care ex vivo methods to improve cancer diagnosis, monitoring and treatment selection. Despite ImmunoPET?s potential, standard antibody-based targeting methods result in long multi-day procedures, insufficient specificity and contrast for early detection, and risk of adverse immunological response with repeated use. While solutions such as minibodies or affibodies can resolve some of these issues, no complete solution to all three of these challenges is available. Aptamers, single-stranded nucleic acid ligands, can yield high binding affinity and specificity to a target, favorable pharmacokinetics, and a highly non- immunogenic composition. However, aptamers have been stymied in clinical imaging due to rapid nuclease-mediated degradation and difficulty retaining affinity in vivo within biological environments, preventing them from accumulating in target tissue in sufficient quantities. Hypothesis: Next-gen aptamer targeting agents, designed with increased longevity and performance within in vivo environments, offer a non-immunogenic alternative to antibody-based targeting agents with the possibility of increased imaging quality and favorable pharmacokinetics. Preliminary Data: Aptitude has developed Chemically-Augmented Particle Display (CAPD), a novel platform that yields next-gen aptamers with unprecedented affinity, specificity, and in vivo stability via a novel non-SELEX approach of ultra-high-throughput quantitative screening. Aims: In this proposal, we aim to utilize CAPD to develop and validate an initial suite of next-gen aptamer-based immunoPET imaging agents, and then compare to established antibody-based methods in an animal model. First, we will use CAPD to create next-gen aptamers to an angiogenesis marker, VEGF, and a checkpoint marker, PD-L1. We will synthetize and conjugate aptamers for radiolabeling, and characterize their affinity, specificity, and stability in human serum. We will validate our 68Ga-labeled anti-VEGF aptamer in vivo, and with an ovarian cancer xenograft model, assess image development time, sensitivity and biodistribution. We will compare to 89Zr-labeled-bevacizumab, the radiolabeled form of Avastin, an FDA-approved anti-VEGF therapy, previously validated in immunoPET imaging. If successful, this study will support both the validation of our aptamer imaging agents and the applicability of our aptamer generation platform, which we plan to use to create a broad suite of safer, faster, and more sensitive in vivo imaging agents for PET.